Fluorescence digital imaging microscopy system

ABSTRACT

A method of preparing cell samples for viable cell number quantification with a fluorescence digital imaging microscopy system employing digital thresholding technique. The cell sample is stained with a first, fluorescent dye and treated with a second dye that is able to quench the fluorescence of the first dye. The fluorescent dye accumulates in viable cells only and is used to stain the viable cells. The second dye is excluded from viable cells but enters non-viable cells, thereby quenching the background fluorescence in non-viable cells and the medium. Two examples of dye combinations are described: fluorescein diacetate used as the fluorescent dye with eosin Y as the quenching dye; and calcein-AM used as the fluorescent dye with trypan blue as the quenching dye. By reducing the background fluorescence, the dynamic range and accuracy of viable cell number measurements are enhanced. In low viability cultures treated with fluorescein diacetate, background fluorescence completely masked viable cells, but digital thresholding and eosin treatment dramatically reduced background fluorescence, producing a linear response over 4 logs of viable cell density.

This is a divisional of application Ser. No. 09/066,134 filed Apr. 24,1998, now U.S. Pat. No. 6,459,805 which is a continuation-in-part ofprior application Ser. No. 08/622,110 filed Mar. 26, 1996, now abandonedwhich applications are hereby incorporated by reference in theirentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a system for quantifying relative cell numbersin tissue culture containers, and more particularly to a system forquantifying relative cell numbers in tissue culture containers usingfluorescence digital imaging microscopy.

2. Description of the Prior Art

Measurement of relative cell numbers (total and/or viable only) isnecessary for a wide variety of biological, immunological, andtherapeutic studies and often requires analyzing many replicate samples.The use of multi-well tissue culture plates, such as the 96-well plates,provides a convenient format for such assays. Microwell assays forrelative cell number have used radioactive isotopes (⁵¹Cr release or ³Hthymidine incorporation), colorimetric substrates produced by viablecells (MTT), or fluorescent dyes that accumulate in viable cells(fluorescein diacetate) or all cells (Hoechst 33342, ethidium bromide,etc.). Assays using radioactive isotopes require specialized wastedisposal and the risk of exposure to radioactivity, while calorimetricassays may provide less precision or dynamic range than isotopic assays.Assay systems using fluorescent dyes are particularly attractive becauseof their ease of use, lack of radioactive waste, short incubation times,and because of the ability to rapidly measure cell numbers directlywithout disrupting cells.

Measuring the fluorescence of a sample involves illuminating the samplewith light of suitable wavelengths, and recording the intensity of thefluorescence produced by the sample. Fluorescence readers that arecurrently commercially available use a photomultiplier tube to directlymeasure total fluorescence in a well, without using a focusing mechanismto measure the fluorescence of defined areas of a well. Sincephotomultiplier tubes detect total fluorescence, they cannotdiscriminate between background fluorescence and intracellularfluorescence. Consequently, assays must rely on rinsing steps toeliminate background fluorescence.

Furthermore, existing fluorescence readers have limited flexibility. Forexample, the Baxter Pandex FCA required the use of custom Pandex platesto wash the cells and an internal bead standard using custom Fluoriconreference beads. Therefore, existing florescence readers offer poorflexibility, low sensitivity and dynamic range, especially whenbackground fluorescence is high.

Another limitation of commercial fluorescence readers and existingmethods for quantification of cell number is the inability todiscriminate between the fluorescence from the viable cells andbackground fluorescence in the wells from dye in the medium and deadcells.

SUMMARY OF THE INVENTION

It is an object of this invention to provide a high degree ofsensitivity and dynamic range for measurements of the number of viablecells.

It is a further object of the invention to provide a high degree ofsensitivity and dynamic range for viable cell number measurements in thepresence of large numbers of non-viable cells.

It is a further object of the invention to create a system forquantifying cellular fluorescence in situ, using a variety of tissueculture plate formats.

In response to these goals, a computer-controlled digital imagingmicroscopy scanning system is developed as described herein. The systemis configured to accommodate a variety of tissue culture plates.Focusing optics are used to form an image of a defined area of theplate, which image is recorded by a recording device and digitized. Thedigital image, in the form of fluorescence light intensity at each pixelof the image, is then manipulated by the computer software to reducebackground noise and extract desired information. The plate is mountedon a motorized stage, the movement of which is controlled by thecomputer. As the stage moves according to the computer commands, aseries of images, or frames, are recorded, the frames covering the wellsto be measured. This is referred to as a scan. During such a scan, themovement of the stage, the recording and digitizing of the images andthe extraction of information occur in a synchronized manner undercomputer control.

The stage is designed to accommodate tissue culture plates of variousformats, and the computer software is designed to control the stagemovement for scanning the plate according to the format inputted to thecomputer. The algorithm for automatic determination of the scanningmovement is described in more detail hereinafter.

The software quantifies fluorescence for the sample according to apredetermined method, described in more detail hereinafter, and sendsthe results both to the computer screen and to a data file. The softwareallows for two types of calibration, and provides easy to use menus forentering configuration, calibration and assay parameters, or dataanalysis specifications.

For the purpose of relative cell number determination, totalfluorescence of a well is obtained by summing the fluorescence lightintensities of all pixels in all frames for the well. The softwareprovides for thresholding ability to enhance signal and reduce noise inthe digital images. For example, fluorescence light intensities that arebelow a predetermined threshold may be rejected by using a Look UpTable. Because intracellular fluorescence is concentrated in a smallarea and is more intense, while background fluorescence is much morediffuse and is less intense, thresholding effectively reduces thebackground noise and increases the dynamic range of the cell numbermeasurement. For samples with high background fluorescence, thistechnique accomplishes cell number measurement in one step, andeliminates the need for extra steps, such as rinsing, which would beotherwise required to reduce background fluorescence.

A method is also described for enhancing the dynamic range and accuracyof viable cell number measurements by treating the sample with a seconddye to quench background fluorescence from the medium and non-viablecells. The first (fluorescent) dye accumulates in viable cells, whilethe second (quenching) dye enters non-viable cells but not viable cells,thereby quenching the fluorescence of the first dye in non-viable cellsand the medium. When fluorescein diacetate (FDA) is used as thefluorescent dye, eosin Y (2′4′5′6′-Tetrabromofluorescein) may be used toquench the background fluorescence. When calcein-AM (Glycine,N,N′-[[3′,6′-bis(acetyloxy)-3-oxospiro[isobenzofuran-1(3H),9′-[9H]xanthene]-2′,7′-diyl]bis(methylene)]bis[N-[2-[(acetyloxy)methoxy]-2-oxoethyl]]-,bis(acetyloxy)methyl]ester) (Molecular Probes, Inc., Eugene, Oreg.) isused as the fluorescent dye, trypan blue(3,3′-[3,3′-Dimethyl[1,1′-biphenyl]-4,4′-diyl)bis(azo)]bis[5-amino-4-hydroxy-2,7-naphthalene-disulfonicacid] tetrasodium salt) may be used to quench the backgroundfluorescence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram that illustrates the overall configuration of thepreferred embodiment of the fluorescence digital imaging microscopysystem of the present invention.

FIG. 2 is the Stage Object Edit screen of the control software of thefluorescence digital imaging microscopy system.

FIG. 3 is the Assay Edit screen of the control software of thefluorescence digital imaging microscopy system.

FIG. 4 illustrates two scan patterns for a well. FIG. 4(A) showsunderscanning, in which all frames remain within the boundaries of eachwell. Some portions of the well are not imaged with this method. FIG.4(B) shows overscanning, in which frames extend beyond the boundaries ofeach well, but image the entire area of the well.

FIG. 5 is a plate scanning diagram which illustrates the direction ofthe scan for a multiwells plate. The overall direction of travel variesdepending on each row. In this example, odd rows are traversed towardsthe right while even rows are traversed to the left.

FIG. 6 is a segment of the software in pseudocode which illustratesoptimization of stage movement and frame summation.

FIG. 7 illustrates image thresholding.

FIG. 8 is the Analysis Menu screen of the control software of thefluorescence digital imaging microscopy system.

FIG. 9 shows the results from example 1, initial serial dilution tests.

FIG. 10 shows the results from example 2, in situ serial dilution tests.

FIGS. 11 and 12 show the results from example 3, digital thresholdingand eosin Y treatment.

FIG. 13 shows the results from example 4, trypan blue treatment of tumorcells stained with calcein-AM.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Hardware

The overall configuration of the preferred embodiment of the presentinvention is illustrated in FIG. 1. An Olympus CK-2 inverted microscope2 is configured for epi-illumination with a 100 Watt mercury vapor lamp4 (HBO-100), fitted for Olympus IMT-style filters. The stage 6 of theinverted microscope 2 has an opening which is modified to fit varioustissue culture plates, including 96-well, 24-well, and 6-well formats(McBain Instruments, Chatsworth, Calif.). Vexta stepper motors 8(Oriental Motor Company, Japan, available from McBain Instruments) aremounted on the stage for providing movements of the stage in twohorizontal directions, the X and Y directions. Stepper motor resolutionis 12,500 steps per revolution, and lead screw pitch is 1.0 mm. A Cohu(San Diego, Calif.) model 4815-2000 monochrome video camera 10 with theAutomatic Gain Control (AGC) jumper disabled is attached to the standardtrinocular head of the inverted microscope 2 with an 80/20 beamsplitter. A KS-1381 image intensifier 12 (VideoScope International,Sterling, Va.) may be installed between the trinocular head and thevideo camera. The above hardware subsystems are readily available fromcommercial sources.

The digital imaging microscopy system of the present invention iscontrolled by a computer 14. In the preferred embodiment, the computersystem comprises a Hewlett-Packard Vectra microcomputer with a 25 Mhz80386 processor running MS-DOS 3.3, 2 megabytes of memory, VGA graphics,Microsoft mouse, and an Ultrasync color monitor (Princeton GraphicSystems, Princeton, N.J.). A PC-Vision imaging board (ImagingTechnology, Woburn, Mass.) is used for video capture with output to aPM970 monochrome monitor 16 (Ikegami, Maywood, N.J.) on the BNC “green”cable. The PC-Vision board provided 512×480 pixel resolution and 256gray levels per pixel with standard video output at 30 frames perminute. Stepper motors are guided by a Compumotor (Parker-Hannifin,Petaluma, Calif.) PC-23 controller board and two Compumotor C-Drivecontrollers 18.

Optical filters are readily available for the standard Olympus IMT mountand are as follows: For Hoechst 33342 either an Olympus “U” IMT cube, oran Omega Optical (Woburn, Mass.) XF05 filter set (excitation 345 nm,emission 475 nm). For FDA and BCECF-AM either an Olympus “B” IMT cube,or an Omega Optical XF22 filter set (excitation 490 nm, emission 525 nm)(22). Omega Optical filters have a special coating to eliminate infraredinterference. For Olympus filters without the special coating, an OmegaOptical BG1 filter is placed in front of the excitation filter on theIMT filter block to eliminate infrared emissions.

Software

In the preferred embodiment, the digital imaging microscopy system iscontrolled by a software developed with Turbo Pascal 6 (Borland, ScottsValley, Calif.). Text mode windows, menus, and dBASE™/FoxPro™ filecompatibility are provided by TOPAZ (Software Sciences, Burlingame,Calif.), a library of screen and database primitives. SigmaPlot™(Jandell Scientific, San Rafael, Calif.) is used to create graphs andcalculate coefficients of correlation. Standard deviation is calculatedby the software.

To provide for greater flexibility, the software program stores systemand assay specific information in data files. Parameters relating tosize, shape, and number of wells are stored in individual “stage object”files. System specific information, such as type of video board, jumpersettings, I/O addresses, and stepper motor specifications, is stored ina configuration file while specifications describing individual assaysare stored in separate assay files. With this generalized design, thesoftware can be quickly adjusted for changes in the hardware.

As described in more details hereinafter, the software automaticallycalculates the proper stage position for all readings, moves the stageand quantifies each frame of fluorescence image in a synchronizedmanner, and quantifies relative fluorescence for each well.

Parameter Input and Operation

The software provides for input of various control parameters necessaryto control the system operation. Tissue culture plate parameters areinputted to the system through the Stage Object Edit screen, as shown inFIG. 2. This screen allows the user to enter the number of wellsvertically and horizontally, the shape of the wells (round or square),the well dimensions (diameter, or sides in mm), the distance betweenwells in mm, and the reference point. The reference point can be anyeasily identified position on the tissue culture plate which can be usedto align the plate in the video monitor. Plates are described to thesystem only once and then become part of a plate selection menu. At thetime an assay definition is created the user simply selects the platefrom a menu of plate choices. Thus, nearly any format of a tissueculture container can be described to the system, including 96-well,24-well, and 6-well plates, individual 35 mm dishes, and 25 cm² flasks.These parameters are stored in individual “stage object” files.

The user enters assay parameters through the Assay Edit Screen, shown inFIG. 3. Specifications are stored in individual assay files. The usercan enter a description for the assay and filenames for both the assayfile and the data output file. When the “Plate” item is highlighted amenu appears allowing selection of a tissue culture plate from a list ofthose which have already been described to the system. If the number ofwells to be scanned is less than the total number of wells in the plate,the user can modify both the number of wells desired and the initialwell to be scanned.

The “Well Scan” item allows for selection of scanning mode: Underscan orOverscan. Underscanning selects the video frames which are completelycontained within each well, even though some areas of the well are notcovered. Overscanning allows every portion of the well to be coveredeven though this results in portions of some frames falling outside theboundaries of the well. FIGS. 4(A) and 4(B) illustrate Underscanning andOverscanning, respectively. Underscanning is generally selected whenspeed is important while Overscanning is selected when it is importantto scan the entire well, for example, to ensure detection of smallnumbers of cells. Switching between Underscanning and Overscanningsimply involves changing the setting on the Assay Edit Screen.

The next item, “Pixel Summation,” controls the way in which video imagesare treated. For example, using the “Total Fluorescence” function, thevalues of all pixels in each video frame are summarized, yielding asingle intensity value for each well scanned. Assay Start Date/Time andCurrent Date/Time may be modified as needed. They are logged to the datafile each time the assay is performed, so results may be taken overmultiple time periods. For this type of assay, results are storedtogether in one data file which can later be used to plot the change influorescence intensity over time.

A “Plate Registration” section also appears on the Assay Edit Screen.This is a descriptive section in which information specific to the typeof tissue culture plate is displayed in a read-only fashion, describingthe proper alignment for the type of plate. Instructions vary fordifferent types of tissue culture plates. For example, 96-well platesmay be marked A1, A2, A3, B1, B2, B3, etc., while 6-well plates arenumbered 1 through 6. The user adjusts the stage manually with the arrowkeys, aligns the plate, and begins the assay. The system then controlsthe stage and scans the plate according to the selections made by theuser.

Occasionally, artifacts appear in a well, such as highly autofluorescentforeign objects. The user can mark these wells as bad data points duringthe assay by pressing the Space bar, and the data point is eliminatedfrom further analysis. Alternatively, individual wells may be excludedfrom analysis after the assay is completed by selecting a special editfunction from the menu.

Calibration

Prior to scanning plates the system is calibrated. There are two typesof calibrations: daily and frame-to-stage calibration. Daily calibrationinvolves adjusting the fluorescence intensity of the microscope throughthe use of standard beads prior to an assay. It consists of adding twodrops of Hoechst beads (Flow Cytometry Standards, Research TrianglePark, N.J.) to 1.0 ml distilled water in a microfuge tube, mixingthoroughly by shaking, and loading into a column (eighth wells) of a96-well plate, 100 μl per well. Wells are scanned and the microscopeaperture diaphragm is adjusted repeatedly until the fluorescenceintensities per well are within the desired range.

Frame-to-stage is performed only when there is some physical change tothe microscope setup, such as changing the magnification, adding orremoving the image intensifier, or changing the type of culture plate tobe analyzed. This calibration involves correlating the image on thescreen, in pixels, to the physical size of the same area on the stage,in millimeters. To perform the calibration, the user places anetched-glass micrometer on the stage so that the image of the micrometerappears on the video monitor. The calibration routine superimposeshorizontal and vertical hatch marks on the image of the micrometer every50 pixels. The user then aligns the micrometer marks with the hatchmarks by moving the stage with the arrow keys and by entering the numberof pixels equal to a specific distance on the micrometer. For both X andY directions, the system automatically calculates pixels per millimeter,and stores this information in the system configuration file.

Scanning Algorithm

Referring to FIG. 5, in the preferred embodiment of the presentinvention, tissue culture plates are scanned with a serpentine, orback-and-forth motion, left and right across rows and up and down withinindividual wells. The scanning pattern for the entire plate isautomatically determined by the system. Based on the calibrationinformation, specifications in the stage object and assay files, and thealgorithm described below, the program calculates the number of videoframes required to scan each well and their position, storing all theinformation needed to control the stage and camera for the entire tissueculture plate in a command file.

In the preferred embodiment, the algorithm for mapping individual videoframes to individual microplate wells is as follows. The diameter of thewell for a round well, or the sides for a rectangular well, are knownfrom the predetermined values in the Stage Object File. The width of thevideo frame is calculated from the well diameter and the calibrationvalue in pixels per millimeter. The system determines the number offrames in the horizontal direction and centers them in the well. Then,for each of the horizonal frames, the system determines the number ofvertical frames required for that particular “slice” of the well andcenters them vertically. FIGS. 4(A) and 4(B) are examples of individualframes mapped to a well. Once all the frame positions for one well aredetermined, the beginning position of the next well is calculated, andthe process repeats for the remaining wells in the plate.

Quantification of Fluorescence

The software quantifies relative cell numbers by analyzing the pixelintensities of the images digitized from the video camera. The PC-Visionboard provides 256 intensity levels for each pixel; the brighter thefluorescence, the higher the numerical value. Total fluorescence perwell should be directly proportional to total cell number when thesupravital DNA-staining dye Hoechst 33342 is used, and to viable cellnumber when FDA or BCECF is used. By summing the pixel intensities forall the video frames covering a well, the software determines the totalrelative fluorescence intensity for the entire well, which isproportional to cell number.

Programming Techniques to Minimize Plate Scanning Time

Two programming techniques are employed to minimize the time forscanning a plate. First, as shown by the pseudocode in FIG. 6, theprogram is organized so that stage movement takes places concurrentlywith the summation of the current video frame, producing a pseudomulti-tasking effect. The software freezes the video frame, issues thestage movement command, and sums the pixels in the frame while the stageis physically in motion.

Second, a more efficient usage of certain Turbo Pascal instructions isdesigned to access memory directly which resulted in much fastersummation of pixels. In the 80386 microchip, memory is addressed byBase, and Offset. The Base Address describes a certain segment inmemory, and the Offset gives a certain offset from that segment. InTurbo Pascal, the specific instruction is: Mem[Base:Offset]. As shown inFIG. 6, in calculating the summation, the Base is incremented while theOffset is kept constant. The Base is incremented by eight in FIG. 6because the absolute offsets, 00_(hex) to 3F_(heX), correspond to eightsegments. This is significantly faster than a simple looping algorithmin which the Base Address remains constant and the Offset isincremented.

Thresholding

Digital thresholding is used to distinguish desired intracellularfluorescence from unwanted background fluorescence. Becauseintracellular fluorescence is concentrated in a small area and is moreintense while background fluorescence is much more diffuse and is lessintense, contribution to the total fluorescence measurement frombackground fluorescence is reduced by ignoring light intensities whichare below a specified value.

In the preferred embodiment of the present invention, thresholding isaccomplished by changing the default values stored in a Look Up Table(LUT) in the imaging hardware within the PC-Vision board. Pixelintensities which enter the hardware are compared against the LUT, andoutput intensities are assigned. By altering the values in the LUT, theoutput intensities can be changed. Referring to FIG, 7, in a normal LUT,every output value is the same as the input value. In a thresholded LUT,output values are mapped to zero for all input values below thethreshold, while output will be the same as input for values above thethreshold. For example, if pixel intensities below 30 representbackground fluorescence, then setting the threshold to 30 will force allpixels with values less than 30 to be zero, effectively decreasingbackground fluorescence without affecting the brighter values.

The software allows the user to select any threshold intensity levelbetween zero and 255, and alters the values in the LUT accordingly. Theuser determines the threshold setting manually by viewing a well on thevideo monitor and adjusting the threshold value (through a menu) to alevel at which the background is visibly black while all cells in thefield are still visibly bright.

While a particular thresholding method is described herein, other formsof digital image manipulation, such as background subtraction, are alsopossible within the capacity of the present hardware and softwareconfiguration.

Analysis

The software provides for data analysis through the Analysis Menu, asshown in FIG. 8. The user can review the raw data file and mark anyinvalid data points which are not marked during the assay by selectingEdit Bad Data Points. In addition, means and standard deviations can becalculated and output in a format suitable for import by other softwareprograms. The user selects the choice which corresponds to the manner inwhich the plate is loaded. For example, a 96-well plate might containsix different conditions such that each condition uses 16 wells in apattern which is two across by eight down. The user would select number1 from the menu, and the software program would calculate the means andstandard deviations for the six groups and write these to the outputfile.

Quenching the Background Fluorescence

The dynamic range and accuracy of viable cell number measurements can beenhanced by quenching the fluorescence in non-viable cells and themedium. Two dyes are used to treat the cell sample. The first is afluorescent dye that accumulates in viable cells only. Although such adye also enters dead cells, it leaks out from the non-viable cells dueto loss of membrane integrity and therefore does not significantlyaccumulate in dead cells. The concentration of the dye in dead cells isno more than two times its concentration in the medium. The first dyemay be, for example, an ester of fluorescein or related dyes which arelipophilic, apolar, and cross the cell membrane. Non-specific esterasesinside the cell hydrolyze the ester dye to a non-ester anionic dye whichis trapped inside the cell if the cell is viable and has an intactmembrane. Since dead cells leak the dye back out, only viable cellsbecome brightly fluorescent.

The second dye used to quench the fluorescence of the first dye is a dyethat enters dead cells but not viable cells. The second (quenching) dyemay be, for example, an acid dye typically used for “dye exclusion”tests, such as trypan blue, eosin Y, Acid Black 2 (a dye in the nigrosinfamily), direct yellow 59 and primuline yellow (primulin). In dyeexclusion tests, these dyes are substantially excluded by viable cellsbut can enter the membranes of dead cells, thereby staining them. Thelevels of such dyes inside viable cells are, for example, at least 1000times lower than their concentrations in the medium. The second dye mayquench the fluorescence of the first dye by, for example, being in closeproximity of the first dye, or binding to the first dye.

EXAMPLES

The examples described below illustrate the performance of the preferredembodiment of the present invention. In examples 1-3, cells were stainedwith fluorescein diacetate (FDA) and treated with eosin Y to quenchbackground fluorescence in non-viable cells. In example 4, cells werestained with calcein-AM and treated with trypan blue to quenchbackground fluorescence.

For examples 1-3, fluorescent dye solutions and assays were prepared asfollows.

Fluorescein diacetate (FDA) (Sigma Chemical Co., St. Louis, Mo.), or2′,7′-bis-(2-carboxyethyl)-5(and-6) carboxyfluorescein, acetoxymethylester (BCECF-AM) (Molecular Probes, Inc., Eugene, Oreg.) was dissolvedin dimethyl sulfoxide (DMSO) to produce a 1 mg/ml stock solution. Thestock was filtered through 0.8 μm nylon, aliquoted into 1.5 ml microfugetubes and stored frozen at −20° C. in the dark. For individual assays,an intermediate stock was prepared by thawing and diluting an aliquotwith RPMI-1640+10% Fetal Calf Serum (FCS) such that adding 50 μl of dyesolution to a well would produce a final concentration of 8 μg/ml. Tominimize reaction with endogenous enzymes in the FCS (which couldincrease the background fluorescence by cleaving ester groups to formfree fluorescein), intermediate dilutions were prepared immediatelyprior to loading each individual plate.

Hoechst 33342 (Calbiochem, San Diego, Calif.) was dissolved in doubledistilled water to produce a 1 mg/ml stock solution, 0.8 μm filtered,and stored in 1.5 ml microfuge tubes in the dark at 4° C. For individualassays, an intermediate stock was prepared by diluting an aliquot withRPMI-1640+10% FCS such that delivery of 50 μl would produce a finalconcentration of 10 μg/ml in the well. Hoechst 33342 was more stablethan FDA and BCECF-AM, and did not need to be mixed separately for eachindividual plate.

The 1.0% (w/v) solution of eosin Y (Sigma Chemical Co., St. Louis, Mo.)in 0.9% NaCl was stored at room temperature.

Cell lines used were the human neuroblastoma cell line SMS-KCNR and theBA-1 hybridoma cell line. Cell lines are grown in a CO₂ incubator inRPMI-1640+10% FCS. SMS-KCNR cells were removed from the tissue cultureflask by washing and incubating in a monolayer of Puck's EDTA for 10minutes. BA-1 cells grow in suspension and were collected bycentrifugation. Cells were then pipetted to break up clumps, centrifugedand counted by hemacytometer and trypan blue exclusion.

Example 1

Initial tests for the linearity and sensitivity of the preferredembodiment of the fluorescence digital imaging microscopy system wereperformed on cells which were treated with dye separate from the 96-wellplate, to minimize any effects from loading dye in situ. BA-1 hybridomacells (which grow as a single cell suspension) were incubated withBCECF-AM in a test tube and rinsed with RPMI-1640+10% FCS prior toloading in a 96-well plate. FIG. 9 shows the serial dilution curve.Fluorescence at 4× magnification was very linear over a range from 340cells/well through 350,000 cells/well (3.4×10³ cells/ml to 3.5×10⁶cells/ml), with a coefficient of linearity equal to 0.995. The systemprovided excellent linearity covering approximately 3 logs offluorescence and 3 logs of cell density.

Example 2

For initial testing of in situ linearity, Hoechst 33342 dye was usedbecause it produces minimal background fluorescence. FIG. 10 shows atypical dilution curve for SMS-KCNR, a neuroblastoma cell line, inRPMI-1640+10% FCS treated in situ with Hoechst 33342 (10 μg/ml for 30min. at 37° C.) in a 96-well plate and scanned at 4× magnificationwithout an image intensifier. Good linearity (r=0.997) was seen from1×10³ cells/well to 5.4×10⁵ cells/well (1.0×10⁴ cells/ml to 5.4×10⁶cells/ml). Results were equally good at magnifications of 10× and 4×with image intensification, producing coefficients of linearity of 0.997and 0.994 respectively.

Example 3 Eosin Y Treatment of Samples Containing Large Numbers ofNon-Viable Cells

Serial dilution of viable cells. For studies of the correlation ofrelative fluorescence and viable neuroblastoma cell number, cells wereloaded in Falcon 96-well tissue culture plates (Becton-Dickinson,Lincoln Park, N.J.) with an Electrapette multi-channel pipettor (MatrixTechnologies, Lowell, Mass.) set for serial twofold dilution mixing 125μl volume in 3 cycles. One million cells per well to two cells per wellwere plated in 8 replicate wells per condition. Final well volume was125 μl. Neuroblastoma cells were allowed to settle and attach for 2-8hours. Prior to scanning, the tissue culture plates were gently loadedby multi-channel pipettor with 50 μl per well of intermediate FDA stockand incubated at 37° C. for 30 minutes. After incubation, plates wereloaded with 30 μl per well of 0.5% eosin Y solution, yielding finaleosin Y concentration of 0.083%; controls received a medium withouteosin Y. Relative fluorescence was determined with the digital imagingmicroscopy system, using 4× magnification with image intensifier andOmega Optical XF22 filters.

Serial dilution of viable cells with additional dead cells. To study theinfluence of large numbers of dead cells on the ability of the digitalimaging microscopy system to detect small numbers of viable cells,identical plates with serial fourfold dilutions of cells were preparedas before. Prior to staining with FDA, half of the plates were loadedwith 4×10⁴ of non-viable cells per well by removing 50 μl of media andreplacing it with 4×10⁴ dead cells resuspended in 50 μl of RPMI-1640with 10% FCS. Non-viable cells for these experiments were obtained fromthe identical cell line, resuspended in RPMI-1640 with 10% FCS andfrozen in 2° C. for 4 hours. Cell viability was determined after thawingby trypan blue dye exclusion counts to be less than 1%.

As shown in FIG. 11a, scans of FDA stained SMS-KCNR neuroblastoma cellsproduced a nearly flat curve with the digital imaging microscopy systemusing no method to reduce the background fluorescence across variouscell concentrations, as fluorescence produced by the viable cells wasobscured by background fluorescence in the medium. We analyzed replicateplates using digital image thresholding, the result of which is shown inFIG. 11b, and this decreased background fluorescence and allowed us todetect varying numbers of viable cells over 3.6 logs of dynamic range.Adding eosin Y to the sample markedly decreased background fluorescencewithout obscuring fluorescence from viable cells if used at a finalconcentration of 0.083%, as shown in FIG. 11c. The combination of boththresholding and eosin Y is shown in FIG. 11d, and the interference ofbackground fluorescence was reduced, and the scan produced a linearincrease in relative fluorescence intensity with increasing numbers ofviable cells over nearly 5 logs. When the FDA concentration was 8 μg/ml,there was tapering off of fluorescence at cell densities above 1×10⁵cells/well, possibly due to exhaustion of the dye (FIGS. 11b, 11 c, 11d). When the FDA concentration was increased to 12 μg/ml, scans wereobtained with excellent linearity covering approximately 5 logs offluorescence intensity and 4 logs of cell density (from 1×10⁶ cells/wellto 1×10² cells/well), as shown in FIG. 11e. The optimal concentration ofFDA was dependent on the cell line used and the highest cellconcentration in an assay, FDA concentrations>12 μg/ml increasedbackground fluorescence, and concentrations from 8-12 μg/ml were foundto be optimal.

FIG. 12 shows the effectiveness of our background reduction method(combination of digital image thresholding and eosin Y addition), when alarge excess of non-viable SK-N-SH neuroblastoma cells was added to theculture plates containing viable SK-N-SH cells prior to staining withFDA. Large numbers of non-viable cells could increase backgroundfluorescence during a cytotoxicity assay by releasing esterases. Bothcontrol and test plates were stained and scanned with the digitalimaging microscopy system under identical conditions. The control platesproduced a linear increase in fluorescence intensity with increasingviable cell number, with the correlation coefficient equal to 0.995, asshown in FIG. 12a. The scans of a replicate plate with 4×10³ additionalnon-viable cells/well produced a comparably linear relationship with thecorrelation coefficient equal to 0.996, and <10 viable cells/well wereeasily detected even in the presence of high numbers of non-viablecells, as shown in FIG. 12b.

Example 4

The performance of the fluorescence digital imaging microscopy systemaccording to the present invention was demonstrated in an antibodydependent cellular cytotoxicity assay (ADCC). In this assay, tumor cellswere stained with calcein-AM, a fluorescent dye similar to FDA. Thetumor cells were mixed with effector cells at various effector to target(E:T) ratios. The effector cells in this case were human neutrophils,armed with a 15 micrograms/ml of anti-GD2 antibody. The monoclonalantibody bound by the fc portion to the fc receptors on the neutrophils,causing them to bind to the tumor cells (target cells) and kill them.Thus, there is a loss of calcein-AM fluorescence from the cells due totarget cell lysis by neutrophils. The amount of killing is measuredusing the fluorescence digital imaging microscopy system, by measuringthe amount of relative fluorescence in the sample (see FIG. 13).

Due to the existence of residual fluorescence in dead cells, the degreeof cell killing is underestimated if the fluorescence from the deadcells is not distinguished from the fluorescence from the target cells.To increase the accuracy of the measurement, trypan blue was added tothe assay to quench the residual fluorescence in dead cells. As shown inFIG. 13, the measured amounts of tumor cell killing are higher (i.e. therelative fluorescence intensity is lower) when trypan blue was added(the dashed curve with square dots) as compared to when no trypan bluewas added (the solid curve with round dots).

The above examples describe using two dyes for staining the viable cellsand for quenching background fluorescence in non-viable cells and themedium. Used with a fluorescence digital imaging microscopy system orother instruments that are suitable for quantifying fluorescence, thismethod enhances the dynamic range and accuracy of the viable cell numbermeasurements. Although two specific dye combinations are described,other combinations may also be used.

In addition, although the chemical compounds are generally described as“dyes,” the word “dye” is not intended to impart any positive limitationon the range of compounds that may be used in the described method,either in terms of chemical structure or in terms of chemical andphysical properties.

It will of course be appreciated by those skilled in the art that thepresent invention is not limited to the precise embodiment disclosed.For example, various changes, alterations and modifications may be madeto the hardware subsystems. It is also understood that although thisapplication refers to multi-well tissue culture plates in describing thepreferred embodiment, the stage and the scanning routine of thedescribed embodiment can be modified to scan tissue culture containersof other shape or format, including individual dishes and flasks.

We claim:
 1. A method for reducing background fluorescence in a culturecell sample during quantification of viable cell numbers usingfluorescent dyes, comprising: treating the sample with eosin Y, whereinthe eosin Y enters non-viable cells but is substantially excluded byviable cells, wherein the background fluorescence in the medium andnon-viable cells in the sample is quenched; and measuring an intensityof fluorescence of the treated sample.
 2. The method of claim 1, whereinthe sample is in a container, wherein the measuring step comprisesrecording a digital image of a defined area of the container using animaging device, the digital image representing fluorescence lightintensities of the defined area.
 3. The method of claim 2, wherein themeasuring step further comprises: holding the container on a stage; andmoving the stage and the imaging device relative to each other in atleast two horizontal directions to image a plurality of areas.
 4. Themethod of claim 3, further comprising quantifying relative cell numbersin the sample.
 5. The method of claim 4, wherein the quantifying stepcomprises manipulating digital images to reduce background fluorescenceintensities in the images and acquiring information relating to relativecell numbers.
 6. A method for reducing background fluorescence in aculture cell sample during quantification of viable cell numbers usingfluorescent dyes, comprising: treating the sample with eosin Y, whereinthe sample is in a container, wherein the eosin Y enters non-viablecells but is substantially excluded by viable cells, wherein thebackground fluorescence in the medium and non-viable cells in the sampleis quenched; measuring an intensity of fluorescence of the treatedsample; and quantifying relative cell numbers in the sample.
 7. Themethod of claim 6, wherein the measuring step comprises: holding thecontainer on a stage; recording a digital image of a defined area of thecontainer, the digital image representing fluorescence light intensitiesof the defined area; moving the stage and an imaging device relative toeach other in at least two horizontal directions to image a plurality ofareas; and controlling the movement of the stage and imaging device andcontrolling the recording of the digital images, wherein the relativemovements between the stage and imaging device and the recording of theimages are performed in a synchronized manner and according to apredetermined scanning pattern.
 8. The method of claim 7, wherein themeasuring step further comprises inputting parameters according to aformat of a container having multiple wells.
 9. The method of claim 8,wherein the quantifying step comprises manipulating the digital imagesto reduce background fluorescence intensities in the images andacquiring information relating to relative cell numbers.